Circ0005512促进雌性大鼠脊髓损伤后小胶质细胞/巨噬细胞焦亡  

Circ0005512 promotes microglia/macrophage pyroptosis after spinal cord injury in female rats

作　　者：张妍[1] 张文凯 张雯秀 刘涛 马子谦 陈学明[1,2] Zhang Yan;Zhang Wenkai;Zhang Wenxiu;Liu Tao;Ma Ziqian;Chen Xueming(Central Laboratory,Beijing Luhe Hospital,Capital Medical University,Beijing 101149,China;Department of Orthopedics,Beijing Luhe Hospital,Capital Medical University,Beijing 101149,China)

机构地区：[1]首都医科大学附属北京潞河医院中心实验室,北京市101149 [2]首都医科大学附属北京潞河医院骨中心,北京市101149

出　　处：《中国组织工程研究》2024年第31期5029-5035,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金青年项目(81901241),项目负责人:张妍。

摘　　要：背景:神经炎症是导致继发性脊髓损伤的重要因素,小胶质细胞/巨噬细胞焦亡是介导脊髓损伤后神经炎症的重要部分,研究显示脊髓损伤后小胶质细胞/巨噬细胞发生焦亡,但circRNA对脊髓损伤后小胶质细胞/巨噬细胞焦亡的调控机制尚不清楚。目的:探讨circ0005512在脊髓损伤后调节小胶质细胞/巨噬细胞焦亡中的作用和机制。方法:将雌性Wistar大鼠随机分为假手术组和脊髓损伤组,BBB评分评估运动功能,苏木精-伊红染色检测脊髓空洞面积,二代测序筛选脊髓组织中差异表达的circRNA,Real-time PCR验证circ0005512的表达。免疫荧光、Western blot、ELISA、Real-time PCR检测脊髓损伤大鼠的细胞焦亡情况以及脂多糖诱导的小胶质细胞系HAPI细胞的焦亡情况,基因敲降验证circRNA0005512对小胶质细胞焦亡的调控作用。结果与结论:①脊髓损伤7 d后,脊髓损伤部位出现明显空洞,脊髓损伤后即刻大鼠运动功能完全缺失,随着时间的推移,脊髓损伤组大鼠运动功能逐渐部分恢复,BBB评分与假手术组比较有显著差异;②二代测序筛选到circ0005512明显上调,且在动物模型和细胞模型中均得到了验证;③免疫荧光、Western blot、Real-time PCR、ELISA实验证实脊髓损伤组织和脂多糖诱导的HAPI细胞中焦亡标志物(NLRP3、GSDMD、caspase-1)均明显上调;④在脂多糖诱导HAPI细胞中敲降circ0005512后,细胞焦亡标志物NLRP3明显下调;⑤以上结果表明circ0005512促进大鼠脊髓损伤后小胶质细胞/巨噬细胞焦亡。BACKGROUND:Neuroinflammation is an important factor leading to secondary spinal cord injury,and microglia/macrophage pyroptosis is a significant part of post-spinal cord injury neuroinflammation.Studies have shown that microglia/macrophage undergoes pyroptosis after spinal cord injury,but the regulatory mechanism of circular RNA(circRNA)in microglia/macrophage pyroptosis after spinal cord injury remains unclear.OBJECTIVE:To investigate the role and mechanism of circRNA0005512 in regulating microglia/macrophage pyroptosis after spinal cord injury.METHODS:Female Wistar rats were divided into sham group and spinal cord injury group.Motor function was evaluated using the Basso,Beattie,and Bresnahan(BBB)scale.Cavity volume was assessed by hematoxylin-eosin staining.Differential expression of circRNA in spinal cord tissue was screened using RNA-sequencing and circ0005512 was validated by real-time PCR.Immunofluorescence,western blot assay,ELISA,and real-time PCR were performed to detect cell pyroptosis in the rats and lipopolysaccharide-induced microglial cell line HAPI cell models.Gene knockdown was used to confirm the regulatory role of circRNA0005512 in microglia/macrophage pyroptosis.RESULTS AND CONCLUSION:(1)Seven days after spinal cord injury,evident cavities were observed at the injury site.Immediately after spinal cord injury,the motor function of rats was completely lost.Over time,the motor function of rats in the spinal cord injury group gradually partially recovered,and there was a significant difference in BBB scores compared to the sham group.(2)Circ0005512 was significantly upregulated according to the results of the RNA-sequencing and confirmed in both the animal and cell models.(3)Immunofluorescence,western blot assay,real-time PCR,and ELISA confirmed the significant upregulation of cell pyroptosis markers(NLRP3,GSDMD,and caspase-1)in spinal cord injury tissue and lipopolysaccharide-induced HAPI cells.(4)In the cell model,knockdown of circ0005512 resulted in significantly decreased levels of cell pyropt

关 键 词：脊髓损伤 circRNA 小胶质细胞 巨噬细胞 焦亡 NLRP3 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R651.2